AIDS, or acquired immunodeficiency syndrome, is caused by human immunodeficiency virus (HIV) and is characterized by several clinical features including wasting syndromes, central nervous system degeneration and profound immunosuppression that results in opportunistic infections and malignancies. HIV is a member of the lentivirus family of animal retroviruses, which include the visna virus of sheep and the bovine, feline, and simian immunodeficiency viruses (SIV). Two closely related types of HIV, designated HIV-1 and HIV-2, have been identified thus far, of which HIV-1 is by far the most common cause of AIDS. However, HIV-2, which differs in genomic structure and antigenicity, causes a similar clinical syndrome.
An infectious HIV particle consists of two identical strands of RNA, each approximately 9.2 kb long, packaged within a core of viral proteins. This core structure is surrounded by a phospholipid bilayer envelope derived from the host cell membrane that also includes virally-encoded membrane proteins (Abbas et al., Cellular and Molecular Immunology, 4th edition, W.B. Saunders Company, 2000, p. 454). The HIV genome has the characteristic 5′-LTR-Gag-Pol-Env-LTR-3′ organization of the retrovirus family. Long terminal repeats (LTRs) at each end of the viral genome serve as binding sites for transcriptional regulatory proteins from the host and regulate viral integration into the host genome, viral gene expression, and viral replication.
The HIV genome encodes several structural regulatory proteins. The Gag gene encodes core structural proteins of the nucleocapsid core and matrix. The Pol gene encodes reverse transcriptase, integrase, and viral protease enzymes required for viral replication. The tat gene encodes a protein that is required for elongation of viral transcripts. The rev gene encodes a protein that promotes the nuclear export of incompletely spliced or unspliced viral RNAs. The Vif gene product enhances the infectivity of viral particles. The vpr gene product promotes the nuclear import of viral DNA and regulates G2 cell cycle arrest. The vpu and nef genes encode proteins that down regulate host cell CD4 expression and enhance release of virus from infected cells. The Env gene encodes the viral envelope glycoprotein that is translated as a 160-kilodalton (kDa) precursor (gp160) and cleaved by a cellular protease to yield the external 120-kDa envelope glycoprotein (gp120) and the transmembrane 41-kDa envelope glycoprotein (gp41), which are required for the infection of cells (Abbas, pp. 454-456).
HIV infection initiates with gp120 on the viral particle binding to the CD4 and chemokine receptor molecules (e.g., CXCR4, CCR5) on the cell membrane of target cells such as CD4+ T-cells, macrophages and dendritic cells. The bound virus fuses with the target cell and reverse transcribes the RNA genome. The resulting viral DNA integrates into the cellular genome, where it directs the production of new viral RNA, and thereby viral proteins and new virions. These virions bud from the infected cell membrane and establish productive infections in other cells. This process also kills the originally infected cell. HIV can also kill cells indirectly because the CD4 receptor on uninfected T-cells has a strong affinity for gp120 expressed on the surface of infected cells. In this case, the uninfected cells bind, via the CD4 receptor-gp120 interaction, to infected cells and fuse to form a syncytium, which cannot survive. Destruction of CD4+ T-lymphocytes, which are critical to immune defense, is a major cause of the progressive immune dysfunction that is the hallmark of AIDS disease progression. The loss of CD4+ T cells seriously impairs the body's ability to fight most invaders, but it has a particularly severe impact on the defenses against viruses, fungi, parasites and certain bacteria, including mycobacteria.
The different isolates of HIV-1 have been classified into three groups: M (main), O (outlier) and N (non-M, non-O). The HIV-1 M group dominates the global HIV pandemic (Gaschen et al., (2002) Science 296: 2354-2360). Since the HIV-1 M group began its expansion in humans roughly 70 years ago (Korber et al., Retroviral Immunology, Pantaleo et al., eds., Humana Press, Totowa, N.J., 2001, pp. 1-31), it has diversified rapidly (Jung et al., (2002) Nature 418: 144). The HIV-1 M group consists of a number of different clades (also known as subtypes) as well as variants resulting from the combination of two or more clades, known as circulating recombinant forms (CRFs). Subtypes are defined as having genomes that are at least 25% unique (AIDS epidemic update, December 2002). Eleven clades have been identified and a letter designates each subtype. When clades combine with each other and are successfully established in the environment, as can occur when an individual is infected with two different HIV subtypes, the resulting virus is known as a CRF. Thus far, roughly 13 CRFs have been identified. HIV-1 clades also exhibit geographical preference. For example, Clade A, the second-most prevalent clade, is prevalent in West Africa, while Clade B is common in Europe, the Americas and Australia. Clade C, the most common subtype, is widespread in southern Africa, India and Ethiopia (AIDS epidemic update, December 2002).
This genetic variability of HIV creates a scientific challenge to vaccine development. HIV-1 is a highly variable virus, for which intra-subtype variation can be as high as 20% and inter-subtype differences can reach 35% of the amino acid sequence (Thomson, M. M. et al (2002) Lancet Infect Dis. 2: 461-471). Although some reports have demonstrated that cross-clade immune responses can be detected (Cao, H. et al. (1997) J. Virol. 71: 8615-8623; Ferrari, G. et al. (1997) Blood 90: 2406-2416; Walker, B. D. et al. (2001) Nat. Immunol. 2: 473-475), other studies conflict (Burrows, S. R. et al. (1992) Eur. J. Immunol. 22: 191-195; McMichael, A. J. et al (2002) Nat. Rev. Immunol. 2: 283-291). Thus, unless clear evidence for very broad cross-clade reactivity becomes available and it is shown that vaccines can induce strong T-cell responses to many epitopes, it is prudent to match the vaccine immunogens to the Clades and/or CRFs in the target population.
Traditional approaches to vaccine development, such as immunization with live attenuated virus, killed virus or viral subunits, are not proving feasible for HIV. For example, in the macaque-SIV model, live attenuated vaccines cause persistent infection, with some macaques developing AIDS. Moreover, it has been difficult to generate effective neutralizing antibodies to clinical isolates of virus. Combinations of traditional and new approaches with novel immunogens designed to elicit humoral and/or cellular immunity may prove necessary and are being actively sought.
With the difficulties encountered for neutralizing antibodies, another approach to HIV vaccine development is to induce cell-mediated immune responses. Such responses are predominantly mediated by cytotoxic T lymphocytes (CTLs). CTLs, also known as CD8+ T-cells, participate in an organism's defense in at least two different ways: by killing virus-infected cells and by secreting a variety of cytokines and chemokines that directly or indirectly contribute to the suppression of virus replication. The induction and maintenance of strong CD8+ T cell responses require “help” provided by CD4+ T-lymphocytes (helper T-cells).
CTLs recognize peptides that originate from both surface and inner structural and nonstructural HIV proteins. Unlike antibodies, they cannot prevent cell-free HIV from infecting host cells. Therefore, the vaccine-induced prophylactic CTLs must act quickly. For that, they may have to be in sufficient numbers, which may or may not require persistent vaccine stimulation or regular re-vaccinations. Preferably, vaccine-induced CTLs should recognize early and/or abundant HIV proteins of the transmitting virus/clade, target multiple CTL epitopes in functionally conserved protein regions to make it difficult for HIV to escape, and kill target cells efficiently.
To induce CTLs, a prime-boost immunization strategy using plasmid DNA encoding an immunogen as a priming immunization, followed by a boosting immunization with a recombinant virus encoding the same immunogen, has demonstrated efficacy to stimulate CD8+ T cell responses in mice (Hanke et al., (1998a) Vaccine 16:439-445; Schneider et al., (1998) Nat. Med. 4: 397-402; Kent et al., (1998) J. Virol. 72:10180-10188). This strategy has been confirmed and extended for non-human primates (Hanke et al, (1999) J. Virol 73:7524-7532; Allen et al., (2000a) J. Immunol. 164: 4968-4978; Amara et al., (2001) Science 292:69-74; Allen et al., (2002) J. Virol. 76:10507-10511; Shiver et al., (2002) Nature 415:331-335) and humans (McConkey et al., (2003) Nat. Med. 9:729-35). WO 98/56919 discloses a prime-boost immunization strategy to generate a CTL-mediated immune response against malarial and other antigens, such as viral and tumor antigens. This immunization strategy uses priming and boosting compositions, which deliver the same CTL epitope in different vectors, where the vector for the boosting composition is a replication-defective poxvirus vector.
Another aspect of vaccine development is to find formulations capable of inducing CTL responses specific for multiple HIV epitopes. Such vaccines could make it relatively difficult for HIV to escape and would have a better chance to suppress HIV replication. Theoretically, several smaller immunogens delivered individually by separate vaccine vectors would be advantageous over one large multigenic protein expressed from a single vector, because the former immunogens may reach separate antigen-presenting cells and each induce at least one immunodominant response (Singh, R. A. et al., (2002) J. Immunol. 168:379-391). With a multigenic protein, unless cross-priming plays a role in immune stimulation, each component is produced by one cell and thus competes with the others for presentation. Hence, a balance is needed between the breadth of elicited immune responses and practicalities of vaccine development and production, the former increasing and the latter decreasing the number of vaccine components.
Yet another aspect of vaccine development is to address HIV variability. First, vaccines could alternate HIV Clades using one protein from each Clade in their formulations. Second, a cocktail of all immunogens derived from the two or three most common HIV Clades could be used, because the immune system has the capacity to respond to many different epitopes. However, as for other vaccine approaches, “immunodominance” of epitopes could narrow the breadth of T cell responses and prevent prophylactic immunity in response to viral infection (Yewdell, J. W. et al. (1999) Ann. Rev. Immunol. 17: 51-88).
During the course of a viral infection, CTL responses develop a predictable bias in their pattern of epitope recognition. A hierarchy of epitope recognition develops, with most of the CTL response targeted to a very limited number of epitopes. This phenomenon is also known as “immunodominance”. Experimental evidence suggests that immunodominance develops as a consequence of many factors, such as the variety of epitope affinities for the relevant cellular receptor, i.e., major histocompatibility complex (MHC) Class I molecule, the various copy numbers of the epitopes produced by the virus, and differences in epitope processing by the host cellular machinery. Therefore, vaccine strategies that can bypass the hierarchy of epitope bias could result in a broad CTL response that provides a protective immune response against viral infection.
With a few exceptions, most of the known CTL epitopes have been identified in chronically infected individuals and responses to these epitopes have heretofore failed to protect against HIV infection. However, other studies have illustrated that the dominant response is not necessarily the most protective (Gallimore, A. et al (1998) J. Exp. Med. 187: 1647-1657). It is therefore a highly desirable advance in the art to develop HIV immunogens based on conserved protein regions, which are, by definition, common to all Clades (Wilson, C. C. et al. (2003) J. Immunol. 171: 5611-5623). Such an immunogen could comprise conserved protein regions that do not necessarily contain epitopes that are naturally processed by HIV-infected cells, but also comprise subdominant or cryptic epitopes that may be protective. An immunogen capable of inducing strong responses against subdominant epitopes could avoid the problem of immunodominance and therefore, induce broad T-cell responses. Further, cross-clade or clade-universal CTL reactivity could allow for less localized geographic use of such an immunogen.
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